Close membrane-membrane proximity induced by Ca(2+)-dependent multivalent binding of synaptotagmin-1 to phospholipids

Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.     

Deficiency of uridine monophosphate synthase (DUMPS) and X-chromosome deletion in fetal mummification in cattle

Ten mummified fetuses were tested for the deficiency of uridine monophosphate synthase (DUMPS), which is known to contribute to the embryonic and fetal mortality in cattle. Genomic DNAs of the mummified fetuses were extracted from tissue samples collected from the mummies and were amplified by GenomiPhi DNA amplification kit. UMPS gene of the mummies was amplified by polymerase chain reaction (PCR) with DUMPS primers. Out of ten mummies examined, two fetuses were heterozygous (carriers) for DUMPS as indicated by the presences of three bands of 89, 53 and 36 bp. Estimated stage of gestation when the death occurred in the two mummies was 3.5 and 2.5 months, respectively. The other fetuses exhibited only two bands of 53 and 36 bp on the polyacrylamide gel indicated that they were normal. On the other hand, all the mummies were sexed using AMX/Y primers. Specific regions of Y and X chromosomes were amplified by PCR using AMX/Y. The expected 280 bp fragment in the female sample and the 280 and 217 bp in the male sample were observed. Nine mummies had a normal X and Y chromosome bands; however, the other mummified fetus exhibited only Y chromosome band, while the constitutive X chromosome fragment was missing. The estimated stage of gestation when the death occurred in this mummified fetus was 100 days. This might be the first report of DUMPS and X-chromosome deletion at the amelogenin gene in bovine-mummified fetuses in Japan.     

Isolation of protoplast from aerial mycelia and fruit-body formation from regenerated mycelia in Pleurotus cornucopiae

We have developed a simple and efficient method for protoplast isolation from Pleurotus cornucopiae. Protoplasts were isolated from aerial mycelia cultured on potato dextrose agar medium without time-consuming propagation in liquid culture. Protoplast yield was significantly increased by means of a decompressing pretreatment of mycelia in enzyme solution and a subsequent enzyme reaction with vibrational mixing. The isolated protoplasts regenerated mycelia and these mycelia formed fruit-bodies without any morphological abnormalities.     

Computational verification of anesthesia effect on temperature variations in rabbit eyes exposed to 2.45 GHz microwave energy

This paper computationally verifies the effect of anesthesia on temperature variations in the rabbit eye due to microwave energy. The main reason for this investigation is that our previous paper suggested a reduction in blood flow due to the administration of anesthesia, resulting in an overestimated temperature increase. However, no quantitative investigation has yet been conducted. The finite-difference time-domain (FDTD) method is used for calculating power absorption and temperature variation in rabbits. For this purpose, we used a computational rabbit phantom, which is comprised of 12 tissues (including 6 eye tissues) with a resolution of 1 mm. Thermal constants of the rabbit were derived by comparing measured and calculated temperatures. For intense microwave exposure to the rabbit eye, time courses of calculated and measured temperatures were in good agreement for cases both with and without the administration of anesthesia. The point to be stressed is that under anesthesia the thermoregulatory response was inactivated and blood flow and basal metabolism was reduced.     

Allogeneic gastric cancer cell-dendritic cell hybrids induce tumor antigen (carcinoembryonic antigen) specific CD8+ T cells

The development of protocols for the ex vivo generation of dendritic cells (DCs) has led to intensive research of their potential use in immunotherapy. Accumulating results show the efficacy of this treatment on melanomas which are highly immunogenic. However, its efficacy remains unclear in other tumors. In this study, allogeneic gastric cancer cell–DC hybrids were used to determine the efficacy of this type of immunotherapy in gastric cancer. Fusion cells of DC and allogeneic gastric cancer cells were generated by polyethylene glycol (PEG) and electrofusion. These hybrids were used to induce tumor associated antigen (TAA) specific cytotoxic T lymphocytes (CTLs). The DCs were successfully fused with the allogeneic gastric cancer cells resulting in hybrid cells. These hybrid cells were functional as antigen-presenting cell because they induced allogeneic CD4+ T cells proliferation. CD8+ T cells stimulated by the MKN-45-DC hybrid cells were able to kill MKN-45 when used for immunization. The CTLs killed another gastric cancer cell line, MKN-1, as well as a melanoma cell line, 888mel, suggesting the recognition of a shared tumor antigen. MKN-45 specific CTLs can recognize carcinoembryonic antigen (CEA), indicating that the killing is due to tumor antigens as well as alloantigens. This approach suggests the possible use of allogeneic gastric cancer cell–DC hybrids in DC based immunotherapy for gastric cancer treatment.     

A molecular "zipper" for microtubules

The dynamics of the microtubule cytoskeleton are controlled by microtubule-associated proteins (MAPs). In this issue, show that Mal3p, the yeast EB1 homolog, belongs to a new class of MAPs that "zipper" up the seam of the microtubule lattice.     

Time-resolved detection of conformational changes in oat phytochrome A: time-dependent diffusion

Conformational changes in oat phytochrome A (phy) in solution after photoexcitation of the red-absorbing form (Pr) were studied in time-domain by the pulsed laser-induced transient grating technique. It was found that the diffusion coefficient (D) of far-red-absorbing form (Pfr) of large phy (1.3 x 10(-11) m(2) s(-1)) is markedly reduced compared with that of Pr (5.8 x 10(-11) m(2) s(-1)). This large reduction indicates that the conformation of Pfr is significantly changed from that of Pr, so that the intermolecular interaction with water molecules increases. This change completes within 1 ms after the photoexcitation. On the other hand, D of Pr of intact phy (4.1 x 10(-11) m(2) s(-1)) first decreases upon photoexcitation to 0.89 x 10(-11) m(2) s(-1) within 1 ms and then gradually increases with a time constant of 100 ms to the value of Pfr, 1.7 x 10(-11) m(2) s(-1). This slower phase suggests that the conformation of the N-terminal region changes with 100 ms to decrease the intermolecular interaction with water after a global change in the large phy region. The increase of D was interpreted in terms of alpha-helix formation in the Pfr form from the random coil structure in the Pr form.     

Kinetic measurement of transient dimerization and dissociation reactions of Arabidopsis phototropin 1 LOV2 domain

Photochemical reaction of a plant blue-light photoreceptor, Arabidopsis phototropin 1-LOV (light-oxygen-voltage sensing) domain 2, was studied with a view to the diffusion coefficients (D) using the pulsed-laser-induced transient grating method. Although the reaction dynamics completes at a rate of several microseconds as long as it is monitored by the absorption change, the diffusion coefficient was found to be time-dependent in a time range of submilliseconds to seconds. The observed signal can be analyzed by the two-state model, which includes the D-value decrease from D of the reactant (9.8 +/- 0.4) x 10(-11) m2/s to D of the product (8.0 +/- 0.4) x 10(-11) m2/s. The D-value of the reactant implies that the dominant form in the ground state of phototropin 1 LOV2 is the monomeric form in a concentration range of 50-200 microM. According to the Stokes-Einstein relationship, the D-change can be explained by a volume increase of 1.8 times. Furthermore, the rate of the D-change was roughly proportional to the concentration of the sample. These two observations indicate that the LOV2 domain transiently forms a dimer upon photoexcitation. When the sample concentration is increased (>180 microM), a new signal component appears within a few milliseconds. This signal represents a D increase from 8.0 x 10(-11) m2/s to 9.8 x 10(-11) m2/s with a time constant of 300 micros. The completely opposite D-change from that observed in a lower concentration, as well as the concentration dependence, implies that a dimer is formed in the ground state in a higher concentration range, even though the fraction of the dimer is still minor in this range. This dimer is photodissociated, with a time constant of 300 micros. This research clearly shows that the time-resolved diffusion measurement is a very powerful tool for detecting spectrally silent association/dissociation processes during chemical reactions. The photoreaction of the LOV2 domain is discussed.     

Protein tyrosine phosphatase receptor type Z is inactivated by ligand-induced oligomerization

Receptor-type protein tyrosine phosphatases (RPTPs) are considered to transduce extracellular signals across the membrane through changes in their PTP activity, however, our understanding of the regulatory mechanism is still limited. Here, we show that pleiotrophin (PTN), a natural ligand for protein tyrosine phosphatase receptor type Z (Ptprz) (also called PTPzeta/RPTPbeta), inactivates Ptprz through oligomerization and increases the tyrosine phosphorylation of substrates for Ptprz, G protein-coupled receptor kinase-interactor 1 (Git1) and membrane associated guanylate kinase, WW and PDZ domain containing 1 (Magi1). Oligomerization of Ptprz by an artificial dimerizer or polyclonal antibodies against its extracellular region also leads to inactivation, indicating that Ptprz is active in the monomeric form and inactivated by ligand-induced oligomerization.     

Misshapen-like kinase 1 (MINK1) is a novel component of striatin-interacting phosphatase and kinase (STRIPAK) and is required for the completion of cytokinesis

Cytokinesis is initiated by constriction of the cleavage furrow and terminated by abscission of the intercellular bridge that connects two separating daughter cells. The complicated processes of cytokinesis are coordinated by phosphorylation and dephosphorylation mediated by protein kinases and phosphatases. Mammalian Misshapen-like kinase 1 (MINK1) is a member of the germinal center kinases and is known to regulate cytoskeletal organization and oncogene-induced cell senescence. To search for novel regulators of cytokinesis, we performed a screen using a library of siRNAs and found that MINK1 was essential for cytokinesis. Time-lapse analysis revealed that MINK1-depleted cells were able to initiate furrowing but that abscission was disrupted. STRN4 (Zinedin) is a regulatory subunit of protein phosphatase 2A (PP2A) and was recently shown to be a component of a novel protein complex called striatin-interacting phosphatase and kinase (STRIPAK). Mass spectrometry analysis showed that MINK1 was a component of STRIPAK and that MINK1 directly interacted with STRN4. Similar to MINK1 depletion, STRN4-knockdown induced multinucleated cells and inhibited the completion of abscission. In addition, STRN4 reduced MINK1 activity in the presence of catalytic and structural subunits of PP2A. Our study identifies a novel regulatory network of protein kinases and phosphatases that regulate the completion of abscission.